(a) Field of the Invention
This invention relates to a biodegradable, biocompatible hydrogel, which can be used for sealants of suppressing the leakage of blood or air during surgical operation, tissue adhesives, anti-adhesive agents and drug delivery carriers, and a method for producing the same.
(b) Description of the Related Art
Fibrin glues and protein-based adhesives have been mostly used for sealants and adhesives for medical purposes, especially surgical operation. However, the fibrin glues are likely to be detached off from surgical sites due to their quite low adhesive force and also may cause the spread of diseases such as virus infections because they are blood products. As the protein-based adhesives, there have been used products (U.S. Pat. No. 5,385,606, product name: Bioglue®) of using albumin (bovine serum albumin) and glutaraldehyde, and products (product name: GRF Glue®) of using a combination of gelatin, resorcinol and formaldehyde, but they may cause infections due to the use of animals-originated proteins and have poor biocompatibility due to toxic problems of aldehydes which are used as a crosslinking agent.
Lately, in order to replace such blood products or protein products, studies of in situ forming hydrogels using synthetic polymers (usually, polyethylene glycol (PEG)) or carbohydrates (usually, dextran) have been vigorously being conducted and some of them have already been released on the market. Depending on the type of crosslinking reactions, there can be radical polymerization reaction, and nucleophilic and electrophilic substitution reaction. The substitution reaction may be further divided into an imine bond, and an amide bond according to the type of bonds. For an example of radical polymerization, a photo-activated polyethylene glycol (PEG) which is being sold under product name, FocalSeal® (Genzyme), is reported to exhibit higher strength than the fibrin sealants, but it requires a light source and a photo-initiator, and it may cause problems in that blood inhibits polymerization reaction by preventing the penetration of light sources, and inconvenience for use. The imine bond is to be generated from the reaction of an aldehyde group and an amine group, and it has been reported that aldehyded dextrans are used as electrophiles and chitosan or aminated polyvinyl alcohols (US 2005/00028930), multi amino PEG (US 2006/0078536, Biomaterials 29 (2008) 4584-4591), and ε-polylysine synthesized through microbe culture (WO 2006/080523) are used as nucleophiles. However, the aldehyded dextrans still have toxicity resultant from aldehyde groups and lack of stability issue because they are readily oxidized and thus lose their reactivity. For amide bonding, there have been widely used methods of activating a carboxylic acid to a succinimidyl ester and then of reacting it with an amine group which is nucleophilic. It is reported that mostly, an activated multicarboxylate PEG ester is used as an electrophile, and collagen (U.S. Pat. No. 5,162,430) and serum albumin (U.S. Pat. No. RE38,827), which are natural proteins, are used as a nucleophile, but they still have concerns about the spread of diseases as described above. Moreover, there has been disclosed a hydrogel by crosslinking reaction of multifunctional PEG activated ester with chitosan or methoxy PEG-conjugated chitosan (U.S. Pat. No. 6,602,952), but since its gelation time is two hours or so, it is not suitable for an in situ crosslinking hydrogel. As synthetic nucleophiles, examples of using multiamino-, or multi mercapto-PEG (U.S. Pat. No. 5,874,500, product name CoSeal®) and examples of using trilysine (U.S. Pat. No. 6,566,406, product name DuraSeal®) have been developed and commercialized. As stated above, numerous examples of using polyethylene glycol with excellent biocompatibility have been reported as a synthetic polymer.
γ-Polyglutamic acid, a γ-polypeptide produced by the amide bond of γ-carboxylic acid and α-amino group of glutamic acid, is a water-soluble, anionic, biodegradable, and biocompatible polymer which is biosynthesized by Bacillus subtilis, a soybean-fermented food microorganism. Attempts are being made to crosslink γ-polyglutamic acid for use as an absorbent or a hydrogel for medical purpose, and typical examples thereof are as follows.
As methods by ionic bonding, Japanese Patent Laid-Open No. 1999-276572 discloses that a polygamma glutamate complex prepared by hydrogenating quaternary amine salts such as chitosan to carboxylic anions of γ-polyglutamates is used for surgical sutures, wound care dressings, anti-adhesive products, and antihemorrhagics.
As examples of crosslinking by chemical bonding using crosslinking agents, Japanese Patent Laid-Open No. 1999-343339 discloses that γ-polyglutamic acid is crosslinked by use of polyepoxy compounds such as diethyleneglycol diglycidyl ether as a crosslinking agent, and it is applied to anti-adhesive agents in WO 2007/132785. However, since the reaction conditions are 40° C. for 48 hours, or 90° C. for 30 min, it is impossible to apply it for the purpose of in situ gelation in medical fields.
Also, it has been reported that water-soluble carbodiimides are used as a condensate for promoting the reaction of carboxyl groups of γ-polyglutamic acid and nucleophiles. Of them, it has been disclosed in Japanese Patent Laid-Open No. 2002-128899 that the fructose with biodegradability, lysine, chitosan, etc. are used as crosslinking compounds and 3-(3-dimethyl aminopropyl)-1-ethyl carbodiimide (EDC) is used as a water-soluble carbodiimide, but this reaction condition is still too long as 24 hours at a room temperature. Furthermore, it has been disclosed in J. Appl. Polym. Sci. 65, pp 1889-1896, 1997 that γ-PGA-EDC precipitates were produced through the reaction of γ-polyglutamic acid and EDC, followed by the addition of 1,3-propane diamine as a crosslinking agent to prepare a hydrogel. However, this condition is also described to let it stand for one day after mixing and its yield is pretty low as 10% or under.
It has been disclosed in Japanese Patent Laid-Open No. 1997-103479 and Biomaterial 19 (1998) 1869-1876 that when gelatin is reacted with succinimidized α-polyglutamic acid as a crosslinking agent, it is gelated within 30 seconds, but this result suggests that γ-polyglutamic acid merely assists the gelation reaction of gelatins which are naturally gelated at a certain temperature around 40° C., and it does not suggest that γ-polyglutamic acid itself becomes a main ingredient of the hydrogels.
Therefore, there are constant needs of hydrogels with a short gelation time, biocompatibility and biodegradability, and having excellent adhesive force and burst strength so as to be preferably applicable to tissue adhesion.